6 research outputs found
Controlling Self-Assembly of Engineered Peptides on Graphite by Rational Mutation
Self-assembly of proteins on surfaces is utilized in many fields to integrate intricate biological structures and diverse functions with engineered materials. Controlling proteins at bio–solid interfaces relies on establishing key correlations between their primary sequences and resulting spatial organizations on substrates. Protein self-assembly, however, remains an engineering challenge. As a novel approach, we demonstrate here that short dodecapeptides selected by phage display are capable of self-assembly on graphite and form long-range-ordered biomolecular nanostructures. Using atomic force microscopy and contact angle studies, we identify three amino acid domains along the primary sequence that steer peptide ordering and lead to nanostructures with uniformly displayed residues. The peptides are further engineered <i>via</i> simple mutations to control fundamental interfacial processes, including initial binding, surface aggregation and growth kinetics, and intermolecular interactions. Tailoring short peptides <i>via</i> their primary sequence offers versatile control over molecular self-assembly, resulting in well-defined surface properties essential in building engineered, chemically rich, bio–solid interfaces
Self-Assembly of Protein Nanofibrils Orchestrates Calcite Step Movement through Selective Nonchiral Interactions
The recognition of atomically distinct surface features by adsorbed biomolecules is central to the formation of surface-templated peptide or protein nanostructures. On mineral surfaces such as calcite, biomolecular recognition of, and self-assembly on, distinct atomic kinks and steps could additionally orchestrate changes to the overall shape and symmetry of a bulk crystal. In this work, we show through <i>in situ</i> atomic force microscopy (AFM) experiments that an acidic 20 kDa cement protein from the barnacle <i>Megabalanus rosa</i> (MRCP20) binds specifically to step edge atoms on {101Ì…4} calcite surfaces, remains bound and further assembles over time to form one-dimensional nanofibrils. Protein nanofibrils are continuous and organized at the nanoscale, exhibiting striations with a period of ca. 45 nm. These fibrils, templated by surface steps of a preferred geometry, in turn selectively dissolve underlying calcite features displaying the same atomic arrangement. To demonstrate this, we expose the protein solution to bare and fibril-associated rhombohedral etch pits to reveal that nanofibrils accelerate only the movement of fibril-forming steps when compared to undecorated steps exposed to the same solution conditions. Calcite mineralized in the presence of MRCP20 results in asymmetric crystals defined by frustrated faces with shared mirror symmetry, suggesting a similar step-selective behavior by MRCP20 in crystal growth. As shown here, selective surface interactions with step edge atoms lead to a cooperative regime of calcite modification, where templated long-range protein nanostructures shape crystals
Static and Time-Resolved Terahertz Measurements of Photoconductivity in Solution-Deposited Ruthenium Dioxide Nanofilms
Thin-film
ruthenium dioxide (RuO<sub>2</sub>) is a promising alternative
material as a conductive electrode in electronic applications because
its rutile crystalline form is metallic and highly conductive. Herein,
a solution-deposition multilayer technique is employed to fabricate
ca. 70 ± 20 nm thick films (nanoskins), and terahertz spectroscopy
is used to determine their photoconductive properties. Upon calcining
at temperatures ranging from 373 to 773 K, nanoskins undergo a transformation
from insulating (localized charge transport) behavior to metallic
behavior. Terahertz time-domain spectroscopy (THz-TDS) indicates that
nanoskins attain maximum static conductivity when calcined at 673
K (σ = 1030 ± 330 S·cm<sup>–1</sup>). Picosecond
time-resolved terahertz spectroscopy using 400 and 800 nm excitation
reveals a transition to metallic behavior when calcined at 523 K.
For calcine temperatures less than 523 K, the conductivity increases
following photoexcitation (Δ<i>E</i> < 0) while
higher calcine temperatures yield films composed of crystalline, rutile
RuO<sub>2</sub> and the conductivity decreases (Δ<i>E</i> > 0) following photoexcitation
Imaging Active Surface Processes in Barnacle Adhesive Interfaces
Surface plasmon resonance
imaging (SPRI) and voltammetry were used
simultaneously to monitor <i>Amphibalanus (=Balanus) amphitrite</i> barnacles reattached and grown on gold-coated glass slides in artificial
seawater. Upon reattachment, SPRI revealed rapid surface adsorption
of material with a higher refractive index than seawater at the barnacle/gold
interface. Over longer time periods, SPRI also revealed secretory
activity around the perimeter of the barnacle along the seawater/gold
interface extending many millimeters beyond the barnacle and varying
in shape and region with time. Ex situ experiments using attenuated
total reflectance infrared (ATR-IR) spectroscopy confirmed that reattachment
of barnacles was accompanied by adsorption of protein to surfaces
on similar time scales as those in the SPRI experiments. Barnacles
were grown through multiple molting cycles. While the initial reattachment
region remained largely unchanged, SPRI revealed the formation of
sets of paired concentric rings having alternately darker/lighter
appearance (corresponding to lower and higher refractive indices,
respectively) at the barnacle/gold interface beneath the region of
new growth. Ex situ experiments coupling the SPRI imaging with optical
and FTIR microscopy revealed that the paired rings coincide with molt
cycles, with the brighter rings associated with regions enriched in
amide moieties. The brighter rings were located just beyond orifices
of cement ducts, consistent with delivery of amide-rich chemistry
from the ducts. The darker rings were associated with newly expanded
cuticle. In situ voltammetry using the SPRI gold substrate as the
working electrode revealed presence of redox active compounds (oxidation
potential approx 0.2 V vs Ag/AgCl) after barnacles were reattached
on surfaces. Redox activity persisted during the reattachment period.
The results reveal surface adsorption processes coupled to the complex
secretory and chemical activity under barnacles as they construct
their adhesive interfaces
Imaging Active Surface Processes in Barnacle Adhesive Interfaces
Surface plasmon resonance
imaging (SPRI) and voltammetry were used
simultaneously to monitor <i>Amphibalanus (=Balanus) amphitrite</i> barnacles reattached and grown on gold-coated glass slides in artificial
seawater. Upon reattachment, SPRI revealed rapid surface adsorption
of material with a higher refractive index than seawater at the barnacle/gold
interface. Over longer time periods, SPRI also revealed secretory
activity around the perimeter of the barnacle along the seawater/gold
interface extending many millimeters beyond the barnacle and varying
in shape and region with time. Ex situ experiments using attenuated
total reflectance infrared (ATR-IR) spectroscopy confirmed that reattachment
of barnacles was accompanied by adsorption of protein to surfaces
on similar time scales as those in the SPRI experiments. Barnacles
were grown through multiple molting cycles. While the initial reattachment
region remained largely unchanged, SPRI revealed the formation of
sets of paired concentric rings having alternately darker/lighter
appearance (corresponding to lower and higher refractive indices,
respectively) at the barnacle/gold interface beneath the region of
new growth. Ex situ experiments coupling the SPRI imaging with optical
and FTIR microscopy revealed that the paired rings coincide with molt
cycles, with the brighter rings associated with regions enriched in
amide moieties. The brighter rings were located just beyond orifices
of cement ducts, consistent with delivery of amide-rich chemistry
from the ducts. The darker rings were associated with newly expanded
cuticle. In situ voltammetry using the SPRI gold substrate as the
working electrode revealed presence of redox active compounds (oxidation
potential approx 0.2 V vs Ag/AgCl) after barnacles were reattached
on surfaces. Redox activity persisted during the reattachment period.
The results reveal surface adsorption processes coupled to the complex
secretory and chemical activity under barnacles as they construct
their adhesive interfaces
Oxidase Activity of the Barnacle Adhesive Interface Involves Peroxide-Dependent Catechol Oxidase and Lysyl Oxidase Enzymes
Oxidases
are found to play a growing role in providing functional chemistry
to marine adhesives for the permanent attachment of macrofouling organisms.
Here, we demonstrate active peroxidase and lysyl oxidase enzymes in
the adhesive layer of adult Amphibalanus amphitrite barnacles through live staining, proteomic analysis, and competitive
enzyme assays on isolated cement. A novel full-length peroxinectin
(AaPxt-1) secreted by barnacles is largely responsible for oxidizing
phenolic chemistries; AaPxt-1 is driven by native hydrogen peroxide
in the adhesive and oxidizes phenolic substrates typically preferred
by phenoloxidases (POX) such as laccase and tyrosinase. A major cement
protein component AaCP43 is found to contain ketone/aldehyde modifications
via 2,4-dinitrophenylhydrazine (DNPH) derivatization, also called
Brady’s reagent, of cement proteins and immunoblotting with
an anti-DNPH antibody. Our work outlines the landscape of molt-related
oxidative pathways exposed to barnacle cement proteins, where ketone-
and aldehyde-forming oxidases use peroxide intermediates to modify
major cement components such as AaCP43